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Objective:To investigate the effect of extracellular vesicles derived from lung tissue on intrapulmonary inflammation and the formation of neutrophil extracellular traps (NETs) in sepsis rats.Methods:Sepsis rat model was established by cecal ligation and puncture (CLP). Collagenase D and DNase I were used to dissociate the lung tissue, the impurities were removed by centrifugation, and finally, the extracellular vesicles (Ti-EVs) derived from lung tissue were separated and extracted by differential ultracentrifugation. Eighteen male SD rats were randomly divided into the sham group, sepsis group and Ti-EVs group: in the Ti-sEV group, a sepsis model was established by CLP, and Ti-EVs suspension was instilled through the airway; rats in the CLP group received CLP, and an equal volume of PBS was instilled through the airway; and rats in the sham group was treated with sham operation. The pathological changes of lung tissue were detected by hematoxylin-eosin (HE) staining after 24 h. The content of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the NETs content in lung tissue.Results:The isolated extracellular vesicles derived from rat lung tissue were observed by transmission electron microscopy as double-layer circular cystic vesicles with particle diameter mainly distributed at 150 nm. Western blot showed positive expression of EVs markers CD9, CD63, and Tsg101. HE staining of lung tissue showed alveolar integrity damage and a large number of inflammatory cells infiltrated in the lung of sepsis rats. Compared with the CLP group, the degree of lung tissue damage was more serious in the Ti-EVs group and the levels of IL-1β, TNF-α and IL-6 in the BALF of rats were significantly increased ( P<0.01). The formation of NETs in the lungs of the rats in the sepsis group and the Ti-EVs group was observed under the laser confocal microscope. Compared with the sepsis group, the fluorescence intensity of NETs in the lung tissues of the Ti-EVs group increased significantly. Conclusions:After enzymatic digestion, differential ultracentrifugation and other treatments, the extracellular vesicles derived from rat lung tissue with high purity can be successfully isolated and extracted. In the process of septic lung injury, extracellular vesicles in lung tissue can aggravate the inflammatory response in the lung and promote the formation of NETs.
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Objective:To analyze the differential expression profile of circRNA and the expression changes of Hippo signaling molecule YAP in renal ischemia-reperfusion injury of mice.Methods:A model of renal IR damage in mice was induced, and serum creatinine (Scr) and urea nitrogen (BUN) concentrations and histological changes of samples were detected to assess renal function and tubular injury. Illumina HiSeq 2500 system was used for high-throughput paired-end sequencing to establish the circRNA expression profile with significant differential expression. Real-time quantitative polymerase chain reaction (qRT-PCR) verified the sequencing results and detected related genes. Gene function (GO) and pathway (KEGG) analysis were performed to predict the biological processes and the major signal pathways involved by differentially expressed circRNAs. The expression level of the main signaling molecule was examined by western blot.Results:Twenty-one distinctly differentially expressed circRNAs ( fold change ≥ 2) were found in IR 24 h kidney tissues compared with the expression in the control groups ( P < 0.05), among which 10 circRNAs were observed to be up-regulated and 11 down-regulated. CircRNA.1100 and circRNA.1122 were randomly (random number) selected for verification by qRT-PCR, and the relative expressions after renal IR 1day were decreased by (0.23±0.016) and (0.36±0.12), respectively, which were highly consistent with the sequencing trends. Analysis of biological functions and pathways showed that differential expression circRNA was significantly enriched in cell cycles, division, growth, apoptosis, death, and Hippo signaling pathways. The Hippo pathway effector molecule YAP protein was significantly up-regulated after renal IR 1day and until the 3rd day of IR. Conclusions:CircRNA may be involved in the regulation of renal IR injury. CircRNA and Hippo pathway may play a key role in the development of renal IR injury.
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Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/ p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0. 5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs) , we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence.
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Humans , Active Transport, Cell Nucleus , Cell Nucleus , Metabolism , Cytoplasm , Metabolism , Fluorescent Antibody Technique , Human Umbilical Vein Endothelial Cells , NF-kappa B p50 Subunit , Metabolism , SoftwareABSTRACT
Objective To investigate the inducing effects of selenium dioxide(SeO2 ) on the apoptosis in human cervical carcino-ma cell line Hela and its influence on the expression of apoptosis-related proteins caspase-3 and P53 .Methods Hela cells were trea-ted with different concentrations of SeO2 for 24 h in vitro ;the morphological changes of Hela cells were observed by the optical mi-croscope;the influence of SeO2 on the cell proliferation and vitality was examined by the MTT assay ;the flow cytometry was em-ployed to detect the cell apoptosis rate ;the expressions of caspase-3 and P53 proteins in Hela cells were determined by the Western blot analysis .Results Under the optical microscopy ,SeO2 generated the obvious influence on the cell growth morphology ,a large number of cells became rounded and shrunken ,and lost the normal form ,while the adherence cell number was evidently decreased and the proliferation was slowed down ;the MTT results showed that SeO2 markedly inhibited the cell proliferation and viability in a dose-dependent manner ,in which ,the cell apoptosis rates induced by the 0 ,1 .875 ,3 .750 ,7 .500 ,15 .000 and 30 .000 μmol/L con-centrations of SeO2 were 3 .12% ,30 .56% ,33 .42% ,37 .50% ,45 .43% and 69 .38% respectively ,which revealing the obviously in-creasing trend;the Western blot assay revealed that SeO2 could up-regulate the caspase-3 and P53 levels ,and reached the peak value at the concentration of 7 .500μmol/L .Conclusion SeO2 could induce the cervical cancer cell apoptosis possibly by up-regulating the expressions of caspase-3 and p53 in Hela cells .
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Objective To investigate the expression changes of microRNAs and VEGF-NOTCH in renal ischemic injury in mice, and to explore the potential mechanism associated with renal angiogenesis.Method Male Balb/c mice were subjected to a standard renal ischemia to induce acute kidney injury (AKI) after 45 min of bilateral renal artery clamping. Following 4 h, 24 h of reperfusion or sham operation, kindey tissues were collected and subjected to detect the expression changes of microRNAs which relatived with angiogenesis and VEGF, Flk-1, Notch1 mRNA by Quantitative Real-time RT-PCR. Flk-1 protein was detected by Western blotting analysis at 24 h and 72 h following Ischemia/Reperfusion(I/R) injury. The expression of CD31 was examined in tissue sections by immunohistochemistry staining, and the microvessels in ischemic region of each group were counted. Results miRNA-210 and miRNA-92a expression increased significantly, with prominent changes at 4 h and 24 h after reperfusion( P ＜ 0.05 ). VEGF and Flk-1 mRNA expression and Flk-1 protein were increased in renal I/R compared with control group respectively (P＜0.05 ).Immunohistochemistry staining results of CD31 showed a significant increase of microvessels in renal ischemic region. Conclusion This study first reported the changes in miRNAs expression in response to kidney I/R in mouse. our results implied that miRNAs may be involved in targeting VEGF-Notch pathway signaling to regulate angiogenesis after renal I/R injury. It provided novel insights into the angiogenesis mechanism of renal ischemic injury.
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ObjectiveTostudythemolecularmechanismoflanthanumonblocking lipopolysaccharide(LPS)-mediated activation of nuclear factor-kappaB (NF-κB)signaling in macrophages.MethodsThe RAW264.7 macrophages were cultured routinely and divided into 4 groups randomly:LaCl3 +LPS group,LPS group,LaCl3 group and control group.The nuclear translocation of p65 protein was detected by immunocytochemistry.Total,cytoplasmic and nuclear proteins were extracted respectively,and then the binding activity of p65 with the target gene was measured by ELISA.Western blot assays were also performed to detect the expression levels of the proteins,including nuclear p65,IκBα and IKK kinase,the phosphorylation status of IκBα and IKK kinase.ResultsLanthanum can block LPS-induced activation of p65 protein through various ways,such as inhibiting its nuclear translocation,reducing its expression in the nuclei and decreasing its binding activity with the target genes.Lanthanum inhibited the degradation of LPS-induced IκBα,but the phosphorylation of LPS-induced IKKβ can not be blocked by lanthanum,nor did the phosphorylation ability of p-IKKβ on IκBα.Conclusion Lanthanum inhibited the degradation of IκBα,nuclei translocation of p65 protein and its binding activity with the target genes,thereby inhibited LPS-induced NFκB activation,which might be one of the inhibition mechanisms of lanthanum on nuclear activation.
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BACKGROUND:Up to date,inbred embryonic stem cells(ESCs) line mainly derived from 129 mouse strain and C57BL/6 strain,occasionally from BALB/c mouse strain,however,few reports concerning ESCs lines derived from Ch inese Kunmingmouse strain.OBJECTIVE:To isolate and culture mouse ESCs of kunming species,additionally,to identify its biological properties.DESIGN,TIME AND SETTING:The experiment with cells as observed subjects was conducted in the institute of Urology,First Affiliated Hospital of Nanchang University from May 2006 to June 2007.MATERIALS:Blastocysts of Kunmin mice with 3.5 days of embryonic age.METHODS:Inner cell mass from 3.5 days embryonic age were isolated from Kunming species mice,cultured on feeder layers of primary mice embryonic fibroblasts,and then the cells were isolated and subsequently cultured.MAIN OUTCOME MEASURES:Colony growth was observed and determined by alkaline phosphatase (AKP) staining;The expression of stage-specific embryonic antigen (SSEA)-1,and cell specific genes of c-Myc,Nanog,Oct3/4 and Sox2 were measured by immunofluorescence and RT-PCR;finally,the ability of differentiation in vitro and in vivo was identified.RESULTS:The ESCs-like colonies presented typical morphological characteristics of ESCs,which was positive to AKP and SSEA-1,and could express several cell specific genes,such as c-Myc,Nanog,Oct3/4 and Sox2,and differentiate into various cell types in vitro and in vivo.CONCLUSION:An ESCs line is successfully dedved from Kunming mice,which has typical biological characteristics of ESCs.